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Regenerationof Inflammation during Skeletal Muscle

Myeloid HIFs Are Dispensable for Resolution

Bendahan and Rémi MounierCuvellier, Claire Latroche, Bénédicte Chazaud, DavidPegan, Jacques R. R. Mathieu, Carole Peyssonnaux, SylvainJulien Gondin, Marine Théret, Guillaume Duhamel, Katarina

http://www.jimmunol.org/content/194/7/3389doi: 10.4049/jimmunol.1401420March 2015;

2015; 194:3389-3399; Prepublished online 6 J Immunol

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The Journal of Immunology

Myeloid HIFs Are Dispensable for Resolution of Inammation during Skeletal Muscle Regeneration

Julien Gondin,* Marine The ´ret,†,‡, x ,{ ,1

Guillaume Duhamel,*,1

Katarina Pegan,‖

Jacques R. R. Mathieu, †,‡, x Carole Peyssonnaux, †,‡, x Sylvain Cuvellier, †,‡, x

Claire Latroche, †,‡, x Béné dicte Chazaud, †,‡, x ,{ David Bendahan,* and Re ´mi Mounier †,‡, x ,{

Besides their role in cellular responses to hypoxia, hypoxia-inducible factors (HIFs) are involved in innate immunity and also haveanti-inammatory (M2) functions, such as resolution of inammation preceding healing. Whereas the rst steps of the inam-matory response are associated with proinammatory (M1) macrophages (MPs), resolution of inammation is associated withanti-inammatory MPs exhibiting an M2 phenotype. This M1 to M2 sequence is observed during postinjury muscle regeneration,which provides an excellent paradigm to study the resolution of sterile inammation. In this study, using in vitro and in vivoapproaches in murine models, we demonstrated that deletion of hif1a or hif2a in MPs has no impact on the acquisition of an M2phenotype. Furthermore, using a multiscale methodological approach, we showed that muscles did not require macrophagic hif1aor hif2a to regenerate. These results indicate that macrophagic HIFs do not play a crucial role during skeletal muscle regeneration

induced by sterile tissue damage. The Journal of Immunology , 2015, 194: 3389–3399.

B esides its role in hypoxia, hypoxia-inducible factor 1(HIF-1) has recently emerged as a major transcriptionalregulator of immunity and inammation (1). HIF-1 a canbe stabilized in normoxia by a number of cytokines, growth fac-tors, and other circulating molecules (2). HIF-1 has been shown tobe involved in the regulation of macrophage (MP) function innormoxic conditions. Indeed, HIF-1 is induced in phagocytes bybacteria even under normal levels of oxygen (3, 4), and the acti-vation of the HIF-1 a pathway supports myeloid cell production of defense factors and improves bactericidal capacity (5). Con-versely, some pathogens interfere with HIF activity to improve

their survival and proliferation (6, 7). Inactivation of HIF-1 a de-creases MP motility, invasiveness, and adhesion, whereas hyper-accumulation of HIF-1 a causes hyperinammation in vivo (8).Proinammatory (M1) stimuli such as LPS or IL-1 induce HIF-1 aexpression in MPs (9–11). Furthermore, levels of HIF-1 a arehigh in inammatory conditions such as rheumatoid arthritis(9). In turn, HIF-1 a promotes the production of inammatorycytokines (TNF- a , IL-1, IL-6, and IL-12) (12). These studiesshowed that HIF-1 a is involved in the rst step of innate immu-nity, which is the M1 activation of MPs. However, recent studieshave proposed that HIF-1 a is also involved in anti-inammatory(M2) functions. These have been reported during acute inam-mation (5, 13, 14) and later stages of the innate immune response.Indeed, Rosenberger et al. (15) showed that HIF-1 stimulates theexpression of netrin, which dampens the inammation by atten-uating neutrophil transendothelial migration (through the synthe-sis and activation of adenosine receptor A2BAR on leukocytes).They have proposed a role of HIF-1 according to an endogenousmechanism, which attenuates the acute inammation during mu-cosal inammation. Moreover, HIF-1 is responsible to the selec-tive induction of broblast integrin b 1 required at time of woundrepair in a model of intestinal submucosa hypoxia (16). In ac-cordance with the prohealing properties of HIF, recent studieshave shown less brotic activity in hepatic brotic models in HIFKO mice (17). Inversely, overexpression of HIF-1 a induces -brosis in white adipose tissue (18). These studies have suggestedthat beyond its classical inammatory action, HIF-1 a (particularlyMP HIF-1a ) may be implied in the resolution of inammationpreceding the healing. Finally, it has been further conrmed thatHIF-2a regulates tumor-associated MP function and is an im-portant immune regulator in these cells by mediating some of theM2 responses (19).

The inammatory response is a spatially and temporally coor-dinated process. Although the rst steps of the inammatory re-sponse are associated with M1 MPs, which secrete microbicidaland M1 compounds, resolution of inammation is associated withM2 MPs exhibiting alternative and/or anti-inammatory pheno-types (20). Both in vitro and in vivo studies have demonstrated that

*Aix-Marseille Universite´, Centre National de la Recherche Scientique, Centre deRésonance Magné tique Biologique et Mé dicale, Unité Mixte de Recherche 7339,13385 Marseille, France; †INSERM, U1016, Institut Cochin, 75014 Paris, France;‡Centre National de la Recherche Scientique, Unité Mixte de Recherche 8104,75014 Paris, France; xUniversité Paris Descartes, Sorbonne Paris Cité , 75006 Paris,France; { Centre National de la Recherche Scientique, Unite´ Mixte de Recherche5534, Centre de Génétique et de Physiologie Molé culaire et Cellulaire, UniversitéClaude Bernard Lyon 1, 69622 Villeurbanne, France; and ‖University of Lujbljana,1000 Ljubljana, Slovenia1M.T. and G.D. contributed equally to this work.

Received for publication June 4, 2014. Accepted for publication January 28, 2015.

This work was supported by Aix Marseille University, Centre National de laRecherche Scientique, INSERM, and Universite ´ Paris Descartes.

J.G. and R.M. designed the experiments, analyzed data, and wrote the manuscript;J.G., M.T., G.D., S.C., K.P., J.R.R.M., C.L., and R.M. performed experiments; and

M.T., G.D., C.P., J.R.R.M., C.L., B.C., and D.B. helped with experimental designs,data analysis, and manuscript writing.

Address correspondence and reprint requests to Julien Gondin, Centre de Re ´sonanceMagné tique Biologique et Mé dicale, Unité Mixte de Recherche, Centre National dela Recherche Scientique 7339, Faculte´ de Médecine de la Timone 27, BoulevardJean Moulin, 13005 Marseille, France. E-mail address: [email protected]

The online version of this article contains supplemental material .

Abbreviations used in this article: l 1, longitudinal diffusivity, rst eigenvalue; l 2 ,axial diffusivity, second eigenvalue; l 3, axial diffusivity, third eigenvalue; ADC,apparent diffusion coefcient; BM, bone marrow; BMDM, BM-derived MP; CSA,cross-sectional area; CTX, cardiotoxin; D0, before; D1, day 1; DTI, diffusion tensorimaging; FA, fractional anisotropy; HIF, hypoxia-inducible factor; HP-1, Hypoxyprobe-1;M1, proinammatory; M2, anti-inammatory; MP, macrophage; MRI, magnetic reso-nance imaging; T 2, transverse relaxation time; TA, tibialis anterior; TE, echo time;WT, wild-type.

Copyright 2015by TheAmericanAssociation of Immunologists, Inc.0022-1767/15/$25.00

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MPs can undergo dynamic transitions between M1 and M2 statesof activation, which is called polarization skewing (20, 21).

After an injury, skeletal muscle regenerates ad integrum due tothe properties of the main adult muscle stem cells (satellite cells).MPs are essential for skeletal muscle regeneration (22, 23). Ef-fectively, when the recruitment of circulating monocytes is totallyprevented during the rst 24 h after injury, muscle regeneration istotally inhibited, with the persistence of necrotic bers until 7 d

after injury (22). Moreover, a series of works using mice decientfor either the chemokine receptor CCR2 or its main ligand CCL2/ MCP1 shows impaired muscle regeneration characterized byformation of fat, decrease in the diameter of new myobers, andreduced capillary number (23). This was associated with a dra-matic decrease of MP inltration in muscle (24–26). Other mo-lecular systems involved in cell migration are required for aproper regeneration because they regulate monocyte/MP entryinto damaged muscle (23). Urokinase-type plasminogen activatoris required for MP chemotaxis and is necessary for MP migrationinto skeletal muscle to ensure an efcient regeneration (27). Al-together, these studies demonstrate that MPs are indispensable forefcient muscle regeneration. Soon after injury, muscle-associatedMPs exhibit an M1 prole and stimulate myogenic precursor pro-liferation. From 24 to 72 h later, these MPs skew into M2 MPs thatstimulate terminal differentiation of myogenic precursor cells,their fusion into myotubes, and the growth of the new regeneratingmyobers (22, 23, 28, 29). Thus, postinjury skeletal muscle re-generation provides an excellent paradigm to study events relatedto the resolution of sterile inammation. Recently, Scheerer et al.(30) claimed that myeloid HIF-1 a is essential for skeletal muscleregeneration, using a model of soft-tissue trauma of the mousehindlimb. However, according to the relevant markers for evalu-ation of skeletal muscle regeneration presented in this study, de-letion of HIF-1 a in MPs induces only a delay in short-term periodof skeletal muscle regeneration (30). Furthermore, the resolutionof inammation, which is essential for efcient skeletal muscle

regeneration (22), was not investigated. Indeed, authors assertedthat MPs showed an M2 phenotype already 24 h after injury basedon the analysis of only two M2 markers, which is not sufcient tocharacterize MP phenotype at this time point (31).

Our general hypothesis was that the macrophagic HIFs playa role during the resolution of inammation occurring duringskeletal muscle regeneration. We hypothesized that HIFs maycontrol the resolution of inammation through endogenous regu-lation despite participation in the promotion of the M1 signalsassociated with the rst steps of muscle remodeling. Despite a highdegree of sequence homology, HIF-1 and HIF-2 have been de-scribed to have nonoverlapping, and even sometimes opposing,roles, particularly regarding inammation. Therefore, in line with

a previous work showing in vitro that HIF-1 a and HIF-2a mRNAare differentially expressed in M1 and M2 MPs (32), we investi-gated whether HIFs play a role at the time of MP phenotypeskewing toward M2 prole and later during muscle regenerationand healing. We originally combined in vitro investigations within vivo multimodal magnetic resonance imaging (MRI), includingtransverse relaxation time (T 2) mapping and diffusion tensor im-aging (DTI), which has been recently introduced for noninvasivequantitative monitoring of the different physiological steps asso-ciated with skeletal muscle injury and regeneration (33, 34).

Materials and Methods Mice

Experiments were conducted on adult male animals (8- to 16-wk-oldanimals) from LysM-CRE +/ 2 :HIF-1a / (5), LysM-CRE +/ 2 :HIF-2a / (19),HIF-2a / , and HIF-1a / (named wild-type [WT] in this article).

Muscle injuries

Muscle injury was rst induced by i.m. injection of cardiotoxin (CTX) intibialis anterior (TA; 12 mM, 50 ml/TA; Latoxan, Valence, France). Skeletalmuscle regeneration after a toxic injury, which causes massive myoberdeath, is a useful model for sterile inammation. This process, whichprovides homogenous damage in the whole muscle (31), is tightly asso-ciated with the inltration of a great number of monocytes/MPs in theregenerating muscle until the end of the regeneration (22). The earlieststage of muscle injury is characterized by a robust inltration of Ly6C/G +

F4/80 low monocytes/MPs (M1 MPs) and Ly6C/G + F4/802

neutrophils to thesite of the injury (22). Rapidly (i.e., between 1 and 3 d after CTX injury),M1 MPs skew into Ly6C/G

2F4/80 high MPs (M2 MPs) (29). In other

words, muscle M2 MPs are generated from M1 MPs, not from Ly6C/G2

monocytes (22, 29). Mice were anesthetized with isourane and wereinjected in the TA muscle. Muscles were harvested for analysis at differenttime points postinjury (1–3, 7, 21 d). We used ischemia-reperfusion asa second model of skeletal muscle injury. In brief, hindlimbs of LysM-CRE+/

2:HIF-1a / , LysM-CRE +/

2:HIF-2 a / , and WT mice have been

submitted to ischemia during 2 h (35) and TA muscles were sampled 21 dafter injury.

Detection of hypoxia

In brief, 2,4, and 8 d after CTX lesion (12 mM, 50 ml/TA; Latoxan, Valence,France), mice were injected with Hypoxyprobe (Hypoxyprobe-1 [HP-1],60 mg/kg), and TA muscles were collected. Single-cell suspensions were

obtained from muscles by enzymatic digestion (collagenase B [RocheDiagnostics] 10 mg/ml and Dispase II [Dutscher Dominique] 2.4 U/ml) forcytometry analysis. Immune cells were isolated by CD45 Ab (anti-mouseCD45 PerCP-Cyanine5.5; eBioscience). Pimonidazole forms adducts withthiol-containing proteins at pO 2 # 10 mm Hg. Adducts were detected withprimary mouse anti–HP-1, and specic staining (i.e., “hypoxia status”) wasevaluated with an anti–HP-1/PE Ab (Mouse Dylight549-MAb; Hypoxy-probe).

Magnetic resonance imaging

MRI measurements were conducted on LysM-CRE +/ 2

:HIF-1a / (n = 7),WT (n = 6), and LysM-CRE +/

2:HIF-2a / (n = 3) mice before (D0) and

1 (D1), 2 (D2), 3 (D3), 7 (D7) and 21 d (D21) after CTX injection. Inves-tigations were performed at 11.75 T on a vertical Bruker Avance 500-MHz/ 89-mm wide-bore imager (Bruker, Ettlingen, Germany), equipped withhigh-performance actively shielded gradients (1 T/m maximum gradient

strength, 9 kT/m/s maximum slew rate) and interfaced with Paravision 5.1.A transmit/receive volume radiofrequency coil (birdcage, diameter Ø = 3cm, homogenous length L = 5 cm; Micro 2.5 Probe; Bruker) was used forimage acquisition. Anim al preparation. Mice were initially anesthetized in an inductionchamber using 2% isourane. Each anesthetized mouse was placed ina home-built cradle, which has been specially designed for the strictlynoninvasive high-eld MRI investigation of the left hindlimb muscles (36).Mice lay supine in the cradle with the head up and were maintained usinga teeth holder and tape at the pelvis level. The left foot was placed ona pedal and the leg was rmly immobilized by a small piece of Teonplaced above the knee joint. Then, the cradle was inserted into the 30-mmcoil with the hindlimb muscles positioned at the magnetic center. Throughouta typical experiment, the anesthesia was maintained under spontaneous res-piration (room air ow ∼ 270 ml/min, regular breathing ∼ 90 breaths/min)using 1.7% isourane (vaporizer Univentor 400 anesthesia unit; Univentor,

Zejtun, Malta). The respiration rate was controlled throughout the experi-ment using a compatible monitoring and gating system (SA Instruments,Stony Brook, NY). MRI sequences. Seven contiguous axial imaging slices (thickness = 1 mm)were selected across the left lower hindlimb based on a set of scout images.T2-weighted images were obtained using a 4-shot spin echo-echo planarimaging sequence with the following parameters: eld of view, 2.0 32.0 cm2; matrix, 128 3 128; slice thickness, 1 mm; number of excitation,15; repetition time, 3500 ms; echo times (TEs), 8–48 ms; acquisitiontime, ∼ 20–30 min. DTI experiments were performed with a Stejskal-Tanner preparation and a segmented (4 shots) spin echo-echo planarimaging readout technique. The following acquisition parameters wereused: diffusion gradient duration ( d) = 5 ms; time between diffusiongradients ( D) = 10 ms; b values = (0 and 450) sec/mm 2 and 12 diffusion-encoding directions. Imaging parameters were: bandwidth = 400 kHz;TR = 3500 ms; TE = 20 ms; number of excitation: 15; eld of view =

2.03

2.0 cm2

; matrix size = 1283

128. The total acquisition time was∼ 45 min. For both T 2-weighted and DTI acquisitions, a fat suppressionmodule was used.

3390 HIFs ARE NOT NECESSARY FOR SKELETAL MUSCLE REGENERATION

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Data processing. T2 maps were generated by tting on a pixel-by-pixelbasis the logarithm of the data to the following linear equation: ln(S(TE)) =ln(S0) 2 TE/T2, where S(TE) is the signal at time = TE and S 0 is theequilibrium magnetization. The DTI reconstruction was performed withthe manufacturer software (ParaVision 5; Bruker), and the correspondingdata were processed using an in-house program developed with InteractiveData Language (Research Systems, Boulder, CO). DTI metrics, includingthe three eigenvalues (longitudinal diffusivity, rst eigenvalue [ l 1], axialdiffusivity, second eigenvalue [ l 2], and axial diffusivity, third eigenvalue[l 3]), fractional anisotropy (FA), and apparent diffusion coefcient (ADC)

were obtained. For both T 2 maps and DTI metrics, a region of interest wasselected around the injured TA muscle. These measurements were doneand averaged for the three slices with the largest sections.

Histological analysis

TA muscles were removed, snap-frozen in nitrogen-chilled isopentane, andkept at 2 80˚C until use. For H&E staining, 8- mm-thick cryosections wereprepared.

Isolation of leukocytes and MPs from muscle

Fascia of the TA muscles was removed. The muscles were minced anddigested in RPMI 1640 medium containing collagenase B 0.2% (RocheDiagnostics GmbH) at 37˚C for 1 h. The resulting homogenate was lteredand cells were counted. CD45 + cells were isolated using magnetic sorting(Miltenyi Biotec) and stained with allophycocyanin-conjugated anti-Gr1(Ly6C/G; eBioscience) and PE-conjugated anti-F4/80 Abs (eBioscience).Cells were analyzed using a FC-500 ow cytometer (Beckman Coulter).Analysis was performed with CXP Cytometer.

MP cell culture

MPs were obtained from bone marrow (BM) precursor cells as previouslydescribed (31). In brief, total BM was obtained from mice by ushingfemurs and tibiae BM with DMEM. Cells were cultured in DMEM me-dium containing 20% FBS and 30% conditioned medium of L929 cell line(enriched in CSF-1) and prepared for 7 d as described previously (37).Purity of differentiated MPs was estimated by ow cytometry after F4/80-PECy7 labeling (eBioscience). MPs were seeded at 50,000 cell/cm 2 for allexperiments and were activated with cytokines to obtain various activationstates: IFN- g (50 ng/ml), IL-4 (10 ng/ml), IL-10 (10 ng/ml) to obtain M1,M2a, and M2c MPs, respectively, in DMEM containing 10% FBS mediumfor 3 d.

Phagocytosis assay

Activated MPs were incubated in culture media (previously described) with20 3 106 GFP-conjugated beads (1- mm diameter; Invitrogen) during 2 h.After three PBS washes, MPs nuclei were labeled with Hoechst (Fluka).Experiments were recorded with a DMRA2 microscope (Leica) connectedto a Coolsnap camera (Photometrics) at 20 3 magnication. The number of phagocytic MPs was calculated using the ImageJ software and expressedas a percentage of total cells. For each condition of each experiment, 12 63 elds have been chosen randomly.

Quantitative RT-PCR

Quantitative RT-PCRs wereperformed as described previously (31). In brief,total RNAs were extracted using the RNeasy Mini Kit (Qiagen). One mi-crogram of total RNA was reverse-transcribed using Superscript II ReverseTranscriptase (Invitrogen). Quantitative PCR was carried out on a Real-Time PCR System (LightCycler 480; Roche Applied Science). Reactionmixtures had a nal volume of 20 ml, consisting of 3 ml cDNA, 10 mlSYBR Green I Master (LightCycler 480), and 0.5 mM primers. After initialdenaturation, amplication was performed at 95˚C (10 s), 60˚C (5 s), 72˚C(10 s) for 45 cycles. Calculation of relative expression was determined bythe LightCycler 480 SW 1.5 software (Roche Applied Science), and foldchange was normalized against cyclophilin-A, a housekeeping gene. Twelvegenes known to be markers of MP polarization ( tnf , il1b , ptgs2 , Nos2 , tgfb1 ,Chia , il10 , arg1 , il4ra , Retnla , Vcam1 , and Chi3l3 ), hif1a (forward, 5 9-TGAGCTTGCTCATCAGTTGC-3 9, reverse, 59-CCATCTGTGCCTTCATCT-CA-3 9), and hif2a (forward, 5 9-TGAGTTGGCTCATGAGTT-GC-3 9,reverse, 5 9-TTGCTGATGTTTTCCGACAG-3 9) were analyzed.

Image analysis

H&E-stained muscle sections were recorded with an E800 microscope

(Nikon) at 203

magnication connected to a camera (QIMAGING). Foreach condition of each experiment, 5 6 1 elds chosen randomly in theentire injured area were counted, representing 355 6 19 myobers.

Cross-sectional area (CSA; expressed in mm2) of regenerating myoberswas quantied using the MetaMorph software (Molecular Devices).

Statistical analyses

All experiments were performed using at least three independent primarycultures or at least three animals for in vivo analyses. Statistical analysesof MRI were performed with the Statistica software version 9 (StatSoft,Tulsa, OK). Normality was checked using a Kolmogorov–Smirnov test.Two-factor (group 3 time) ANOVAs with repeated measures on timewere used to compare T 2 values and DTI metrics. When a main effect ora signicant interaction was found, Newman–Keuls post hoc analysis wasused. Statistical analyses of in vivo and in vitro studies were performedwith Student t test. Signicance was accepted when p , 0.05.

Results Deletion of macrophagic HIFs does not affect skeletal muscleinammation and regeneration

To determine whether the tissue injured with CTX was hypoxic, weused pimonidazole labeling (30). Eleven, 67, and 55% of CD45 +

cells extracted from regenerating WT muscle were hypoxic 2, 4,and 8 d after injection of CTX in the TA muscle, respectively(Supplemental Fig. 1 ).

Multimodal MRI investigations, including T 2 mapping and DTI,were performed to investigate in vivo the effects of myeloid HIFsin muscle damage and regeneration. In brief, recent studiesdemonstrated that T 2 changes can be used as an index of muscleinammation (33, 34) even though other biological phenomena(edema, necrosis) can also affect T 2 values (38). In addition,diffusion-weighted MRI provides information about the watermolecules self-diffusion, which is restricted by physical barrierssuch as membranes, cytoskeleton, mitochondria, and sarcoplasmicreticulum, thereby leading to anisotropic diffusion. Diffusion in-dices including the mean ADC, eigenvalues ( l 1, l 2, and l 3), anddiffusion anisotropy (FA) can be derived from DTI. l 1 representsdiffusive transport along the long axis of a muscle ber (39),whereas l 2 and l 3 have been proposed to reect to the long and

short cross-sectional axes of the muscle bers (40), respectively.A signicant time effect ( p , 0.001) was observed for all the

recorded variables indicating that both T 2 values and DTI metricswere signicantly modied as a result of CTX injection for allthree groups. Indeed, T 2 values were signicantly elevated atD1 (+101 6 12%), peaked between D2 and D3 (+139 6 15 and +131 6 15%, respectively), remained elevated at D7 (+36 6 9%),and recovered at D21 (Fig. 1A).

The time course of DTI changes was clearly different from thathighlighted by the T 2 values. l 2, l 3, and ADC values peaked at D1(+37 6 6, +56 6 9, and +32 6 6%, respectively) and remainedelevated at D2 (+32 6 8, +52 6 12, and +29 6 8%, respectively)and D3 (+9 6 9, +20 6 12, and +8 6 8%, respectively) as

compared with values obtained at D0 (Fig. 1B and Table I).These parameters were further reduced at D7 ( 2 12 6 4, 2 13 6 5,and 2 9 6 5%, respectively) and returned to the baseline values atD21. The FA evolved in an opposite way with a signicant re-duction at D1 ( 2 52 6 6%), D2 (2 53 6 5%), and D3 (2 27 610%), an increase at D7 (+18 6 8%), and a full recovery at D21(Table I). l 1 only increased at D1 (+15 6 6%) and D2 (+12 67%), and returned to the baseline values at D3 (Table I).

Importantly, it should be noted that both DTI and T 2 changeswere similar for WT, LysM-CRE +/

2:HIF-1 a / , and LysM-CRE +/

2:

HIF-2a / mice, indicating that macrophagic HIFs are not in-volved in the CTX-induced MRI changes.

HIF-1 a2 / 2 MPs acquire M2 phenotype in vivo

To determine the specic contribution of MP-derived HIF-1 a 2 / 2 ,we performed loss-of-function experiments by using LysM-CRE +/

2:

The Journal of Immunology 3391

http://www.jimmunol.org/lookup/suppl/doi:10.4049/jimmunol.1401420/-/DCSupplementalhttp://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/http://www.jimmunol.org/lookup/suppl/doi:10.4049/jimmunol.1401420/-/DCSupplemental

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FIGURE 1. Multimodal MRI investigations of the effects of myeloid HIF a 1 and HIFa 2 in muscle regeneration. ( A ) Typical representative axial T 2 mapsfrom WT and LysM-CRE +/

2:HIF-1a / mouse hindlimb muscles ( top panels ) and quantitative analysis of T 2 values (bottom panel ) obtained from WT ( n =

6), LysM-CRE +/ 2

:HIF-1a / (n = 7), and LysM-CRE +/ 2

:HIF-2a / (n = 3) mouse TA muscles before (D0) and 1 (D1), 2 (D2), 3 (D3), 7 (D7), and 21 d(D21) after injury. Values are presented as mean 6 SD. (B ) Typical representative axial maps of the third eigenvalue ( l 3) from WT and LysM-CRE

+/ 2 :HIF-1a / mouse hindlimb muscles ( top panels ) and quantitative analysis of l 3 values (bottom panel ) obtained from WT ( n = 6), LysM-CRE +/

2:HIF-1a / (n =

7), and LysM-CRE +/ 2

:HIF-2a / (n = 3) mouse TA muscles at the same respective time points than those mentioned in ( A ). Values are presented as mean 6SD. Signicantly different from values recorded at D0: *** p , 0.001.



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HIF-1a / (5). The capacity of HIF-1 a2 / 2 MPs to switch their

phenotype was assessed in vivo during normal skeletal muscleregeneration. Thanks to ow cytometry analysis coupled withLy6C/G and F4/80 labelings, cell population analysis was per-formed among CD45 + cells extracted from regenerating WT andHIF-1a

2 / 2 muscles at different time points (Fig. 2A). First, thenumber of inltrating neutrophils and MPs was similar in WT andHIF-1a

2 / 2 muscles 1 d after injury (Fig. 2A), excluding a differ-ence of leukodiapedesis of WT and HIF-1 a

2 / 2 myeloid cells.Second, the distribution of the two MPs subsets did not differbetween WT and HIF-1 a

2 / 2 regenerating muscles during the MPskewing period in this model of sterile inammation (i.e., D1, D2,and D3 after CTX injury) (31), showing that LysM-CRE +/

2:HIF-1a /

animals had an unaltered M2 MP development (Fig. 2B). Thus,these results show that in absence of HIF-1 a , M1 MPs were ableto skew in M2 MPs at the time of resolution of inammation.

HIF-1 a2 / 2 MPs acquire M2 phenotype in vitro

Next, BM-derived MPs (BMDMs) from WT and LysM-CRE +/ 2 :HIF-1a / animals were analyzed for their ability to acquire po-larized M1 and M2 phenotypes. BMDMs were polarized withcytokines to trigger various inammatory proles: IFN- g (M1state), IL-4 (alternative state [M2a]), and IL-10 (anti-inammatorystate [M2c]) (41). As we have previously described (31), resultsshow that M1 polarization was achieved in these in vitro con-ditions. Indeed, mRNAs of the M1 markers ( Tnf , il1b , ptgs2 , Nos2 )were strongly expressed in M1 WT MPs (Fig. 3A). However, high

variations were observed among primary cultures for the expres-sion of the M2 markers mRNAs ( Tgfb1 , il10 , il4ra , Vcam1 , Chia , Arg1 , Retnla , Chi3l3 ) (Fig. 3A), although a strong tendency toincrease some of these markers was observed in WT M2 versus M1MPs (Chia , Arg1 , Retnla , Chi3l3 ; Fig. 3A). As a whole, no sig-nicant difference of marker expression was observed between WTand HIF-1 a

2 / 2 MPs (Fig. 3A). Interestingly, expression of HIF-1 amRNA signicantly decreased with the acquisition of M2 pheno-type in WT MPs (Fig. 3B), whereas HIF-2 a mRNA was signi-cantly increased when MPs were polarized toward M2 proles inboth WT and HIF-1 a

2 / 2 MPs (Fig. 3B). Then we evaluated thephagocytic capacity of MPs because this mechanism participates inthe M1 to M2 transition of MPs (22, 31). Thus, we assessed thecapacity of WT and HIF-1 a

2 / 2 MPs to phagocyte GFP-conjugatedbeads. No signicant difference in the percentage of phagocyticMPs was observed in M1, M2a, and M2c between WT and

HIF-1a2 / 2 MPs (Fig. 3C). These results indicate that HIF-1 a is

dispensable for phagocytosis of beads by MPs.

HIF-2 a2 / 2 MPs acquire M2 phenotype in vivo

HIF-1a and HIF-2a display unique and sometimes opposing ac-tivities in regulating cellular functions in both physiological andpathophysiological context (19, 42, 43). As we did for HIF-1 a

2 / 2

MPs, loss-of-function experiments were performed by usingLysM-CRE +/ 2 :HIF-2 a / (19). Thus, cell population analysis wasperformed among CD45 + cells extracted from regenerating WTand HIF-2 a

2 / 2 muscles at different time points (Fig. 4A) to de-termine the capacity of HIF-2 a

2 / 2 MPs to switch their phenotypein vivo. The number of inltrating neutrophils and MPs wassimilar in WT and HIF-2 a

2 / 2 muscles 1 d after injury (Fig. 4A).

Moreover, the distribution of the two MPs subsets did not varybetween WT and HIF-2 a

2 / 2 regenerating muscles during the MPskewing period, suggesting that the acquisition of M2 phenotypeby MPs was unaffected in LysM-CRE +/

2:HIF-2 a / animals

(Fig. 4B). Thus, these results show that in the absence of HIF-2 a ,M1 MPs were able to skew into M2 MPs at the time of resolutionof inammation.

Macrophagic HIFs are not required for proper skeletal muscleregeneration

MPs are necessary for skeletal muscle regeneration, as inhibit-ing monocyte/MP inltration impairs muscle regeneration (22).Twenty-one days after CTX injury, visual histological examina-

tion has shown the same pattern of muscle regeneration in micedecient for HIF-1 a 2 / 2 or for HIF-2 a 2 / 2 in myeloid cells, ascompared with WT mice (Fig. 5A). Furthermore, CSA of the newmyobers (an indicator of skeletal muscle regeneration efciency)in WT, LysM-CRE +/

2:HIF-1 a / , and LysM-CRE +/

2:HIF-2 a /

mice was similar at D21 (Fig. 5B), which is in accordance withour MRI ndings. Finally, distribution of myober CSA of WTmice paralleled distribution of myober CSA of LysM-CRE +/ 2 :HIF-1a / and LysM-CRE +/

2:HIF-2 a / mice (Fig. 5C). Finally,

ischemia-reperfusion was used as another model of sterile skeletalmuscle injury to clarify the role of macrophagic HIFs in skeletalmuscle regeneration. We showed that CSAs of the new myoberswere similar among WT, LysM-CRE +/

2:HIF-1 a / , and LysM-

CRE+/ 2 :HIF-2 a / mice at day 21 (Fig. 5D), considered as a latetime point of skeletal muscle regeneration. Therefore, deletion of HIF-1a or HIF-2a in myeloid cells did not alter skeletal muscle

Table I. The rst two eigenvalues ( l 1 and l 2), ADC, and FA obtained from WT, LysM-CRE +/ 2

:HIF-1a / , and LysM-CRE +/ 2

:HIF-2a / mice before(i.e., D0) and 1 (D1), 2 (D2), 3 (D3), 7 (D7), and 21 d (D21) after injury

D0 D1 D2 D3 D7 D21

l 1 (3 102 3 mm2 /sec)

LysM-CRE +/ 2

:HIF-1a / 1.80 6 0.07 2.08 6 0.03*** 2.05 6 0.04*** 1.80 6 0.10 1.75 6 0.05 1.72 6 0.06WT 1.77 6 0.04 2.09 6 0.07*** 2.04 6 0.05*** 1.84 6 0.05 1.72 6 0.10 1.76 6 0.04LysM-CRE +/

2:HIF-2a / 1.79 6 0.05 1.92 6 0.05*** 1.86 6 0.09*** 1.73 6 0.09 1.59 6 0.01 1.69 6 0.07

l 2 (3 102 3 mm2 /sec)

LysM-CRE+/ 2

:HIF-1a/

1.33 6 0.06 1.84 6 0.03*** 1.77 6 0.05*** 1.41 6 0.10*** 1.18 6 0.06*** 1.30 6 0.03WT 1.30 6 0.02 1.82 6 0.04*** 1.76 6 0.06*** 1.47 6 0.06*** 1.17 6 0.04*** 1.31 6 0.06LysM-CRE +/

2:HIF-2a / 1.30 6 0.02 1.67 6 0.05*** 1.58 6 0.12*** 1.36 6 0.12*** 1.08 6 0.01*** 1.24 6 0.05

ADC (3 102 3 mm2 /sec)

LysM-CRE +/ 2

:HIF-1a / 1.39 6 0.05 1.83 6 0.03*** 1.81 6 0.04*** 1.46 6 0.10*** 1.28 6 0.05*** 1.34 6 0.03WT 1.36 6 0.03 1.84 6 0.03*** 1.80 6 0.05*** 1.53 6 0.06*** 1.25 6 0.06*** 1.35 6 0.04LysM-CRE +/

2:HIF-2a / 1.36 6 0.01 1.69 6 0.06*** 1.61 6 0.12*** 1.42 6 0.12*** 1.16 6 0.02*** 1.30 6 0.05

FALysM-CRE +/

2:HIF-1a / 0.28 6 0.02 0.13 6 0.01*** 0.13 6 0.01*** 0.22 6 0.03*** 0.33 6 0.02*** 0.27 6 0.03

WT 0.28 6 0.01 0.14 6 0.02*** 0.13 6 0.01*** 0.19 6 0.01*** 0.34 6 0.02*** 0.29 6 0.02LysM-CRE +/

2:HIF-2a / 0.29 6 0.02 0.14 6 0.01*** 0.16 6 0.01*** 0.21 6 0.04*** 0.34 6 0.02*** 0.29 6 0.01

*** p , 0.001, signicantly different from D0.



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regeneration, at least in two different models of sterile injury(CTX and ischemia-reperfusion).

DiscussionThe results of this study show that the absence of HIFs in MPs hasno impact on the resolution of inammation in two sterile modelsof skeletal muscle regeneration. Based on the earlier ndings, weconclude that the same types of MPs were generated in the samenumber from an identical cell inltrate in WT, LysM-CRE +/

2:

HIF-1a / , and LysM-CRE +/ 2

:HIF-2 a / muscles during the earlystages of regeneration. In other words, the inltration, differenti-ation, and kinetics of M1 and M2 muscle MPs were found to beidentical in both examined mice strains. Moreover, HIF-1 a

2 / 2

and HIF-2 a 2 / 2 MPs showed no functional alterations in regen-erating muscle. Therefore, although HIF-1 a has been reported to

inuence MP polarization in various tissues (44–46), HIF-decientMPs are able to acquire M2 phenotype and functions in sterile in-ammation during skeletal muscle regeneration. Phagocytosis of tissue debris is a crucial function of MPs in the resolution of in-ammation in skeletal muscle (22, 28). It has been shown previ-ously that HIF-1 a has an important role in phagocytic function of MPs in various experimental models (47, 48) that seems not to bethe case in the present in vitro experiments.

Besides its role in hypoxia and in inammation under normoxicconditions, HIF-1 is involved in skeletal muscle homeostasis andphysiology (43, 49–52). Recently, it has been claimed that mye-loid HIF-1 a was essential for skeletal muscle regeneration aftera soft trauma (30). Our results, showing no alteration of skeletalmuscle regeneration in mice depleted for myeloid HIFs in twodifferent models of sterile injury dispute this assertion. Discrep-

FIGURE 2. HIF-1a2 / 2 macrophages acquire M2 phenotype in vivo. CD45 + cells were isolated from muscle using magnetic sorting and stained with

allophycocyanin-conjugated anti-Gr1 (Ly6C/G) and PE-conjugated anti-F4/80 Abs. ( A ) Number of MPs (Ly-6C/G +F4/80+ and Ly-6C/G2

F4/80+ cells) andneutrophils (Ly-6C/G +F4/80

2cells) was evaluated per milligram of muscle 1 d after the injury from WT and LysM-CRE +/

2:HIF-1a / mouse. ( B ) The

presence of M1 (Ly-6C/G +F4/80+) and M2 (Ly-6C/G2

F4/80+) MPs subsets was expressed as a percentage of total MPs (F4/80 + cells) in muscle at days 1,2, and 3 after injury. Results are means 6 SEM. All experiments were performed using at least three animals.



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FIGURE 3. HIF-1a2 / 2 macrophages acquire M2 phenotype in vitro. ( A and B ) Analysis of M1 markers, M2 markers, and hif1a and hif2a gene ex-

pression in WT and HIF-1 a2 / 2 MPs after in vitro polarization. MPs were obtained from BM precursor cells (BMDMs). BMDMs were activated with

cytokines to obtain various activation states: IFN- g , IL-4, IL-10 to obtain M1, M2a (alternative), and M2c (anti-inammatory) MPs, respectively, for 3 d.(C ) WT and HIF-1 a

2 / 2 MPs were polarized as in ( A ) and (B), incubated in culture media with 20 3 106 GFP-conjugated beads during 2 h. After three PBSwashing, MPs nuclei were labeled with Hoechst. The number of phagocytic MPs was expressed as a percentage of total cells. All experiments wereperformed using at least three independent primary cultures. Signicantly different from WT: *** p , 0.001, * p , 0.05. Signicantly different from M1:### p , 0.001, ## p , 0.01, # p , 0.05. Signicantly different from M2c: $ p , 0.05.



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ancies between the two studies might be related to the model of skeletal muscle injury and the techniques used to quantify myeloidcells (immunohistology versus ow cytometry) and myoberparameters (different number of myobers taken into consider-ation for CSA calculation). Nevertheless, several data appearing inScheerer et al.’s (30) study strongly dampen the title assertion of “an essential” role of macrophagic HIFs in skeletal muscle re-generation. First, the analysis of CSA showed a signicant dif-ference between WT and HIF-1 a

2 / 2 mice only at day 7 after theinjury, whereas no signicant difference was observed at 10 d.This is characteristic of a transient delay at the onset of regener-ation process in this model of injury, discordant with an “essen-tial” function of HIF in myeloid cells. Accordingly, the variationsobserved in myeloid cells between the two mice strains were

observed only between days 2 and 7. To assess a denitive role of HIF1 in myeloid cells, further analysis at later time points of re-generation (i.e., 3 wk) is required (31). Moreover, the study byScheerer et al. (30) reported no alteration of myeloid HIF-1 adeletion on MP inltration or on MP polarization, which is inagreement with the data presented in this article in two models of sterile inammation. Scheerer et al.’s (30) study reported a tran-sient delay in the number of F4/80 + cells, suggesting a defect inthe proliferation of some MPs in the HIF1 a KO strain. Because wedid not observe such a delay in the two models we used andknowing that only M2 Ly6Cneg MPs are capable of proliferation(22), further investigations are required to understand which en-vironmental changes the myotraumatic model is inducing on thesecells. In conclusion, taking in consideration the fact that the pre-

FIGURE 4. HIF-2a2 / 2 MPs acquire M2 phenotype in vivo. CD45 + cells were isolated from muscle using magnetic sorting and stained with allo-

phycocyanin-conjugated anti-Gr1 (Ly6C/G) and PE-conjugated anti-F4/80 Abs. Cells were analyzed using a ow cytometer. ( A ) Number of MPs (Ly-6C/ G+F4/80+ and Ly-6C/G -F4/80+ cells) and neutrophils (Ly-6C/G +F4/80 - cells) was evaluated per milligram of muscle 1 d after the injury from WT andLysM-CRE +/

2:HIF-2a / mouse. ( B ) The presence of M1 (Ly-6C/G +F4/80+) and M2 (Ly-6C/G

2F4/80+) MPs subsets was expressed as a percentage of

total MPs (F4/80 + cells) in muscle at days 1 and 2 after injury. Results are means 6 SEM. All experiments were performed using at least three independentprimary cultures or at least three animals.



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vious work done by Scheerer et al. (30) was carried out in a dif-ferent model of muscle injury, complementary investigations areneeded to determine the discrepancies between the differentmodels of injuries.

Multimodal MRI investigations were performed to assesswhether myeloid HIFs play a role during muscle regeneration. Wefound that the time course of both T 2 and DTI changes resultingfrom CTX injection was similar among WT, LysM-CRE +/

2:HIF-

1a / , and LysM-CRE +/ 2

:HIF-2 a / mice, indicating that bothinammation and muscle regeneration were not delayed in mye-loid HIF-1 a and HIF-2a knockout mice. These ndings clearly

showed that myeloid HIFs are not essential for skeletal muscleregeneration. Although the CTX-induced MRI changes observedin this study are in line with those recently reported in the liter-ature (33), we further demonstrated that the increase of FA oc-curring 7 d after injury (33) was actually related to the decreasedl 2 and l 3 values, thereby reecting the well-known presence of small-diameter regenerating muscle bers (22). Although it hasbeen suggested that T 2 variations represent a marker of muscleinammation (33, 34), it should be pointed out that a linear re-lationship of T 2 and intracellular volume has been previously re-ported (53). On that basis and considering the reduced second and

FIGURE 5. Deletion of myeloid HIFs did not affect skeletal muscle regeneration resulting from an acute injury. ( A ) H&E staining of regenerating musclein WT, LysM-CRE +/

2:HIF-1a / , and LysM-CRE +/

2:HIF-2a / mice. TA muscles were injured with CTX and analyzed 21 d postinjury. TA muscles were

removed, snap-frozen in nitrogen-chilled isopentane, and kept at 2 80˚C until use. Scale bar, 50 mm. Mean ( B ) and distribution ( C ) of myober CSA.Signicantly different between WT and LysM-HIF-1 a

2 / 2 mice, * p , 0.05. Signicantly different between WT and LysM-HIF-2 a2 / 2 mice, $ p , 0.05. All

experiments were performed using at least three animals. ( D ) Macrophagic HIFs are not required for proper skeletal muscle regeneration after injury due toischemia-reperfusion. CSA of the new myobers of TA is calculated 21 d after the injury in LysM-CRE +/

2:HIF-1a / LysM-CRE +/

2:HIF-2a / and WT

mice. All experiments were performed using at least three animals. *** p , 0.001, signicantly different from Nonischemic muscles.



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third eigenvalues at day 7, the associated higher T 2 values mightactually be related to the expansion of the extracellular compo-nent, including, for instance, a widened interstitial space (54, 55),rather than to intracellular changes. Overall, the combination and/ or comparison of multimodal MRI with the recently introducedbioluminescence imaging technique (56) would be of interest formonitoring noninvasively and quantitatively the pathophysiologi-cal events, such as chronic inammation, which are associated

with skeletal muscle diseases.In conclusion, our multiscale methodological approach clearlyshowed that myeloid HIFs are not essential for skeletal muscleregeneration after a sterile injury in two different models.

AcknowledgmentsWe thank Christophe Vilmen (Aix-Marseille Universite ´, Centre National dela Recherche Scientique, Centre de Re ´sonance Magné tique Biologique etMédicale, Unité Mixte de Recherche 7339) for excellent technical assis-tance and Alexandre Boissonnas and Christophe Combadie `re (SorbonneUniversité s, UPMC Université Paris 06, CR7, Centre d’Immunologieet des Maladies Infectieuses, INSERM U1135, Centre National de laRecherche Scientique ERL) for providing the Ccr2

2 / 2 mouse model.

DisclosuresThe authors have no nancial conicts of interest.

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